U1 small nuclear ribonucleoprotein and splicing inhibition by the Rous sarcoma virus negative regulator of splicing element

Retroviruses require both spliced and unspliced RNA for replication, Accumu lation of unspliced Rous sarcoma virus RNA is facilitated in part by a nega tive cis element in the gag region, termed the negative regulator of splici ng (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns, The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoprotein s (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathwa y, yet splicing does not normally occur from the NRS. A mutation that aboli shes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, u sing specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is func tional when paired with a minor-class 3' ss from the human P120 gene, Surpr isingly, the expectation that the same NRS mutants would be defective for s plicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS, Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) oth er than U11. Our data suggest that this factor is U1 snRNP and that a U1 bi nding site that overlaps the U11 site is also disrupted by RG11, Analysis o f mutations which selectively disrupted U1 or U11 binding indicated that sp licing inhibition by the NRS correlates most strongly with U1 snRNP. Additi onally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS, These data indicate a functional role for U1 in NRS-mediated splicing inhibition.